TRD3. Peptide inhibitors and small molecule drug discovery - Gonen (Lead) Summary The discovery, design, and synthesis of small molecule drugs and peptide inhibitors is slow and inefficient as it requires large amounts of material for successful structural characterization. Over the past 50 years, NMR and other powerful spectroscopic techniques were developed to address this challenge. While almost all of them rely on inference of atomic connectivity, the unambiguous determination of a small molecule's structure requires use of X-ray and/or neutron diffraction methods. In reality, X-ray crystallography is rarely applied in such cases for routine drug development. This is because of the significant effort is required for performing crystallization assays and because of the large quantity of material that is needed, while specimens are often available only in femtogram amounts. We recently demonstrated that microcrystal electron diffraction (MicroED) can be used as a powerful and potentially routine method for unambiguous structural determination of small molecules. Using seemingly amorphous powders and peptides straight off of a purification column without further crystallization, MicroED delivered atomic resolution structures using only femtogram amounts of material. The process took minutes and required little personnel effort. Here, we will establish MicroED as a method of choice for structure determination of small molecules and as an analytical tool for drug development through high-throughput pipelines for small molecule structure determination. We will extend the use of MicroED to structure determination of natural products for drug discovery efforts and study neurotoxins and nerve agents that typically block ion channels. The aims are: 1. Real-time structure determination and high-throughput drug discovery; 2. Evaluation of MicroED methodologies' applicability to studying natural products; 3. Neurotoxins, nerve agents, and identification of neutralizing agents. Overall, we will develop procedures for sample preparation for small molecules, natural products and toxins, and deliver a large library of their atomic resolution structures with the long-term goal of aiding drug discovery process.